Neuroactive Chondroitin Sulfate Glycomimetics

Neuroactive Chondroitin Sulfate Glycomimetics

Manish Rawat, Cristal I. Gama, John B. Matson, and Linda C. Hsieh-Wilson*Division of Chemistry and Chemical Engineering and Howard Hughes Medical Institute, CaliforniaInstitute of Technology, Pasadena, California 91125

Chondroitin sulfate (CS) glycosaminoglycans play critical roles in various physiologicalprocesses, including cell division, inflammation, and spinal cord injury.1 However, thecomplexity of this important class of molecules has hindered efforts to relate structure tofunction and to generate defined molecular tools for manipulating CS activity. Comprising40–200 sulfated disaccharide units, CS is thought to contain “blocks” of high and lowsulfation, with highly sulfated regions serving as binding sites for growth factors, cytokines,and other proteins.2,3 Although a tetrasaccharide is often sufficient for molecularrecognition and neuronal activity,3,4 longer sequences may further enhance protein bindingand allow for the assembly of multimeric protein complexes.5 Given the challenges inherentin the synthesis of large oligosaccharides, we sought the development of CS mimetics thatwould retain key properties of CS polysaccharides, yet be more synthetically accessible.Here, we describe the first synthesis and characterization of CS glycopolymers with definedsulfation sequence and tunable chemical and biological properties.

Surprisingly few polymers based on glycosaminoglycan structures have been reported.Heparin-like glycopolymers have been synthesized from simple monosaccharides such as N-acetyl-D-glucosamine.6 However, no high molecular weight glycopolymers have beenassembled from disaccharide building blocks found in heparin/heparan sulfate, chondroitin,dermatan, or keratan sulfate glycosaminoglycans.7 We envisioned that such glycopolymerswould more closely mimic the natural polysaccharides (Figure 1), facilitating explorationsinto the importance of macromolecular structure (e.g., distance, number, and orientationbetween protein-binding epitopes, multivalency) and providing tools to perturb the functionsof CS in vivo.

The functional complexity of CS represents a major challenge to the synthesis ofglycopolymers as hydroxyl, carboxylic acid, and sulfate groups are reactive under cationicand anionic polymerizations. As such, we explored ruthenium-catalyzed ring-openingmetathesis polymerization (ROMP),8 which has been shown to tolerate a broad range offunctional groups and affords glycopolymers of controllable length and narrowpolydispersity.9 In place of norbornene derivatives used previously,9 we chose cis-cyclooctene monomers, as less constrained scaffolds have been reported to adapt better tobiological receptors.10 To ensure a high degree of control over the sulfation pattern, sulfatedmonomers were used in the polymerization reaction.

We first probed the efficiency of ROMP using disaccharides 6 and 7, which display thebiologically active CS-E sulfation motif (Scheme 1). To obtain 6 and 7,trichloroacetimidate11 donor 14 was coupled to the cyclooctene acceptor 2 to affordexclusively the β-linked disaccharide 3 in 69% yield. Removal of the tert-butyldimethylsilyl

[email protected] Information Available: Experimental procedures and compound characterizations. This material is available free of chargevia the Internet at http://pubs.acs.org.

NIH Public AccessAuthor ManuscriptJ Am Chem Soc. Author manuscript; available in PMC 2011 February 7.

Published in final edited form as:J Am Chem Soc. 2008 March 12; 130(10): 2959–2961. doi:10.1021/ja709993p.

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group using HF·pyridine gave 4, which served as a glycosyl acceptor to assembletetrasaccharides (Scheme 2). Free radical reduction of the N-trichloroacetyl group and DDQoxidation of the p-methoxybenzylidene acetal of 3 furnished diol 5 in 82% yield, which gave6 upon sulfation with sulfur trioxide · trimethylamine complex. Alternatively, desilylationfollowed by saponification using a sequential LiOOH–NaOH treatment to minimize β-elimination at the C4 position12 gave deprotected monomer 7.

ROMP is typically performed in non-coordinating, aprotic solvents. However, the lowsolubility of 7 under such conditions prompted us to investigate the reaction in polar proticsolvents. Polymerization of 7 in MeOH using 2.5 mol % of the fast-initiating catalyst(H2IMes)(Py)2(Cl)2Ru=CHPh (8)9g,13 proceeded to incomplete conversion (36%) andfurnished glycopolymer 9 with a low degree of polymerization (DP = 21; Scheme 2).Similarly, ROMP of 7 in aqueous solution using 2.5 or 5.0 mol % of the water-solublecatalyst (H2IMes-poly(ethyleneglycol))(Cl)2Ru=CH(o-iPrOC6H4)14 afforded incompleteconversion (7 or 60%, respectively) and low molecular weight polymers (DP = 8 or 28,respectively). The low conversions and polymer molecular weights are likely due tocoordination of the solvent to the catalyst and reactive intermediates, which is expected toreduce the propagation rate and lead to premature chain termination.13b

To address this problem, we explored the polymerization of disaccharide 6, which wassoluble in MeOH/(CH2Cl)2 cosolvent mixtures. Reaction of 6 with 2.5 mol % of catalyst 8in 1:5 MeOH/(CH2Cl)2 at 55 °C afforded complete conversion to glycopolymer 10 with 40repeating units (Scheme 2 and Table 1, entry 2). Importantly, varying the amount of catalyst(0.5–5.0 mol %) led to a series of glycopolymers with controllable molecular weights (DP =25–80) and relatively narrow polydispersities (1.24–1.63; entries 1–4). To probe theimportance of sulfation, glycopolymer 11, whose pendant disaccharides lack sulfate groups,was also synthesized via polymerization of 5 (entry 5). Finally, we generated glycopolymer13 containing CS-E tetrasaccharide units from monomer 12 (entry 6). To our knowledge, 13represents the most functionally complex glycopolymer synthesized to date. Allglycopolymers were characterized by 1H NMR spectroscopy and gel permeationchromatography (GPC). The desired CS glycomimetics 14–16 were obtained afterdesilylation and sequential LiOOH–NaOH treatment in 87–95% yield over two steps(Scheme 3).

We evaluated the biological activity of the glycopolymers by measuring their ability tomodulate the outgrowth of hippocampal neurons. Neurons were cultured on poly-DL-ornithine-coated glass coverslips, and each glycopolymer was added in solution to theneurons.15 After 48 h, the neurons were fixed, immunostained with anti-tubulin antibodies,and examined by fluorescence microscopy. We found that natural polysaccharides enrichedin the CS-E sulfation motif completely inhibited neurite outgrowth when added in solutionat 0.5 μg/mL glucuronic acid concentration to hippocampal neurons (Figure 2). These resultsare consistent with the model that CS-E present on cell-surface proteoglycans or coated on asubstratum recruits growth factors to the cell surface, thereby stimulating downstreamsignaling pathways involved in neuronal growth.3b,16 By adding CS-E in solution toneurons, growth factors are presumably sequestered away from the cell surface, resulting inneurite inhibition.

Remarkably, glycopolymer 14 mimicked the activity of the natural polysaccharide (Figure2). While the isolated disaccharide 7 was insufficient for biological activity, incorporation ofthe disaccharide into a polymeric framework endowed it with the ability to inhibit neuronalgrowth. Moreover, the potency of the glycopolymers was valence-dependent: polymers with25 disaccharide units exhibited moderate activity (40.9 ± 5.4% inhibition), while those with80 disaccharide units showed significantly enhanced activity (86.0 ± 5.8% inhibition) at the

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J Am Chem Soc. Author manuscript; available in PMC 2011 February 7.

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same glucuronic acid concentration. These findings highlight the importance ofmultivalency in modulating the activity of CS. In addition, the unsulfated glycopolymer 15had little effect on neurite outgrowth (5.9 ± 5.6% inhibition), confirming earlier observationsthat sulfation is a prerequisite for activity,4 and further highlighting the ability of theseglycopolymers to recapitulate features of natural CS polysaccharides.

Previous studies have shown that a tetrasaccharide represents a minimum functional domainfor protein recognition and neuronal growth-promoting activity when adhered to asubstratum.3,4 In this solution-based assay, a monovalent tetrasaccharide at 0.5 μg/mLglucuronic acid concentration had minimal activity (Supporting Information). In contrast,glycopolymer 16 at the same glucuronic acid concentration exhibited maximal activity,inducing neurite inhibition to the same extent as the natural polysaccharide (Figure 2). Tocompare the relative potencies of glycopolymer 16 and the natural polysaccharide, wemeasured their inhibition values at various concentrations (Figure 3). We were excited tofind that the inhibitory potency of 16 was comparable to that of the natural polysaccharide(IC50 values of 1.3 ± 0.1 and 1.2 ± 0.1 nM, respectively), despite considerable changes tothe macromolecular structure. These results suggest a surprising flexibility in the wayproteins engage CS. We find that a linear orientation of CS epitopes is not required forbiological activity. Instead, strategic placement of short epitopes, even in the context ofbranched structures, is sufficient to recapitulate many of the properties of natural CSglycosaminoglycans.

In summary, we have generated novel CS glycomimetics through ROMP of fully protected,sulfated monomers. The polymers recapitulate key features of glycosaminoglycans anddisplay biological activities comparable to those of natural CS polysaccharides. Notably, thisapproach greatly simplifies the synthesis of complex glycosaminoglycans, providingsynthetically accessible, bioactive structures of programmable sulfation sequence. Theability to control the sulfation pattern within the glycopolymer is significant as it shouldallow for the generation of CS type-specific polymers (e.g., CS–E, CS–A, CS–C) withdistinct functions. Our studies highlight the importance of multivalent interactions inamplifying CS activity and suggest an unexpected tolerance for unnatural polymericarchitectures. We anticipate that these glycopolymers will be powerful tools for furtherexploring how the macromolecular structure of CS directs its activity and for manipulatingthe functions of CS in vivo.

Supplementary MaterialRefer to Web version on PubMed Central for supplementary material.

AcknowledgmentsWe thank Prof. Robert H. Grubbs, Dr. Jason P. Jordon, and Dr. Erin Guidry for generous donation of the water-soluble catalyst and for helpful discussions. This work was supported by the American Cancer Society (GrantRSG-05-106-01-CDD), Tobacco-Related Disease Research Program (14RT-0034), and the Howard HughesMedical Institute.

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Figure 1.Schematic representation of (A) natural CS and (B) CS glycopolymers. Grey and whitecircles represent the monosaccharide subunits, red circles represent sulfation.

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Figure 2.CS-E glycopolymers inhibit the outgrowth of hippocampal neurons. (A)Immunofluorescence images of neurons 48 h after treatment with the indicated compound.A fixed glucuronic acid concentration of 0.5 μg/mL was used in each case to examine theeffects of multivalency. The molar concentrations were 14.3 nM for the natural CS-Epolysaccharide, 12.1 nM for the CS-E tetrasaccharide polymer, and 16.6–53.0 nM for thedisaccharide polymers. See Supporting Information for details. (B) Statistical analysis ofneurite length. Error bars represent SEM from three separate experiments. P values arerelative to the untreated neurons. See Supporting Information for conditions.

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Figure 3.Comparison of the inhibitory potencies of CS glycopolymer 16 and the naturalpolysaccharide at various concentrations. IC50 values are based on the molar concentrationof compound required to inhibit neurite outgrowth by 50% relative to untreated neurons. SeeSupporting Information for details.

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Scheme 1. Synthesis of the Disaccharide Monomeraa Conditions: (a) TMSOTf, CH2Cl2, −20 °C, 69%. (b) HF·pyr, THF/pyr, 25 °C, 95%. (c)AIBN, Bu3SnH, benzene, 50 °C, 92%. (d) DDQ, CH3CN/H2O, 25 °C, 89%. (e) SO3·TMA,DMF, 80 °C, 85%. (f) (i) HF·pyr, THF/pyr, 25 °C; (ii) LiOH, H2O2, then NaOH, MeOH, 25°C, 89% (two steps).

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Scheme 2. Synthesis of Protected CS Glycopolymers aa Conditions: (a) 8, 1:5 MeOH/(CH2Cl)2, 55 °C, 24 h. (b) TMSOTf, 4, CH2Cl2, −20 °C,45%. (c) AIBN, Bu3SnH, benzene, 50 °C, 89%. (d) DDQ, CH3CN/H2O, 25 °C. (e)SO3·TMA, DMF, 80 °C, 71% (two steps).

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Scheme 3. CS Glycomimetics aa Conditions: (a) (i) HF·pyr, THF/pyr, 25 °C; (ii) LiOH, H2O2, then NaOH, MeOH, 25 °C,87–97% (two steps).

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Tabl

e 1

RO

MP

of C

S M

onom

ers U

sing

Cat

alys

t 8

entr

ym

onom

erm

ol %

cata

lyst

poly

mer

% y

ield

n (D

P)M

na (g

/mol

)PD

Ia

16

5.0

1088

2528

320

1.63

26

2.5

1059

4043

780

1.34

36

1.0

1055

5863

400

1.35

46

0.5

1053

8087

540

1.24

55

2.5

1169

3936

100

1.61

612

2.0

1351

6411

9 00

01.

33

a Num

ber a

vera

ge m

olec

ular

wei

ght a

nd p

olyd

ispe

rsity

inde

x w

ere

dete

rmin

ed b

y G

PC (0

.2 M

LiB

r in

DM

F).


Neuroactive Chondroitin Sulfate Glycomimetics

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